Anthocyanin Synthesis and the Expression Patterns of Bhlh Transcription Factor Family During Development of the Chinese Jujube Fruit (Ziziphus Jujuba Mill.)

Article Anthocyanin Synthesis and the Expression Patterns of bHLH Transcription Factor Family During Development of the Chinese Jujube Fruit (Ziziphus Jujuba Mill.) Qianqian Shi 1,2, Xi Li 1,2, Jiangtao Du 1,2 and Xingang Li 1,2,3,* 1 College of Forestry, Northwest Agriculture and Forestry University , Yangling District, 712100 Shaanxi, China; [email protected] (Q.S.); [email protected] (X.L.); [email protected] (T.D.) 2 Key Comprehensive Laboratory of Forestry of Shaanxi Province, Northwest A&F University, Yangling District, 712100 Shaanxi, China 3 Research Center for Jujube Engineering and Technology of State Forestry Administration, Northwest A&F University, Yangling District, 712100 Shaanxi, China * Correspondence: [email protected]; Tel.: +86‐29‐87082556 Received: 28 February 2019; Accepted: 17 April 2019; Published: 19 April 2019 Abstract: The basic helix–loop–helix (bHLH) family is an important transcription factor for eukaryotes and is involved in a wide range of biological activities. Among these, bHLH can interaction with WD repeat (WD40 or WDR) and V‐myb avian myeloblastosis viral oncogene homolog (MYB) form a ternary complex to promote the efficient synthesis of anthocyanins. In this study, a total of 138 jujube bHLH (ZjbHLH) family members were screened from the transcriptome of the two jujube cultivars, ‘Junzao’ (JZ) and ‘Tailihong’ (TLH). Of these, 95 ZjbHLH genes were mapped to 12 chromosomes. A phylogenetic tree was constructed using 27 arabidopsis bHLH (AtbHLH) protein sequences of Arabidopsis thaliana (L.) Heynh. and 138 ZjbHLH protein sequences of jujube. The results show that the ZjbHLH family of jujube can be divided into 12 subfamilies. The three candidate genes, ZjGL3a, ZjGL3b and ZjTT8, related to anthocyanin synthesis, were classified into subgroup III. Meanwhile, ZjGL3a, ZjGL3b and ZjTT8 have high homology with the bHLH transcription factors involved in anthocyanin synthesis in other plants. In addition, it was found that the jujube ZjbHLH transcript family showed changing patterns of expression during fruit development. The relative expression levels of ZjGL3a, ZjGL3 and ZjTT8 were consistent with the changes of the anthocyanin contents in the two jujube cultivars examined. To better understand the anthocyanin synthesis pathway involved in ZjbHLH, a regulatory pathway model for anthocyanin synthesis was constructed. This model involves the processes of anthocyanin signal transduction, synthesis and transport. Keywords: ZjbHLH transcription factor; jujube; anthocyanin; anthocyanin synthesis pathway 1. Introduction Chinese jujube (Ziziphus jujuba Mill.) belongs to the Rhamnaceae and is indigenous to China. It has been domesticated in China as a fruit crop for more than 7000 years [1]. This species is in high demand, due to its rich flavor and high nutritional value, in many Asian countries [2]. In more recent years, human nutritional science has demonstrated that jujube is a rich source of vitamins, flavonoids, polyphenols, fiber, sugars and other beneficial components [3]. Anthocyanins are widely distributed in the fruits, flowers, seeds and other tissues of the jujube plant. Here, they serve as antioxidants, attract insect pollinators, resist ultraviolet rays and participate in the plant’s responses to biotic and Forests 2019, 10, 346; doi:10.3390/f10040346 www.mdpi.com/journal/forests Forests 2019, 10, 346 2 of 12 abiotic stresses [4]. The synthesis of anthocyanins is closely related to the flavonoid synthesis pathway. Sequence studies of different plant species have shown that the anthocyanin synthesis gene is strongly conserved, and also that the flavonoid synthesis gene is especially conservative [5]. The basic helix–loop–helix (bHLH) family of transcription factors (named for its basic‐helix‐ loop‐helix structure) is large and widely distributed in eukaryotes. Its core conserved domain contains approximately 60 amino acid residues [6]. The bHLH has been shown to be involved in numerous biological processes, including stress resistance [7–9], growth and development [10,11], signal transduction [12,13] and synthesis [14–16]. The first bHLH transcription factor was discovered in maize, where its function is involved in the synthesis of anthocyanins [17]. Other family members have subsequently been found in several species, including Arabidopsis [18], tobacco [19], rice [20] and tomato [21]. At this stage, 150 arabidopsis bHLH (AtbHLH) family members have been identified from Arabidopsis and have been shown to be involved in a range of processes, including the development of the reproductive system, transduction of phytochrome signaling, synthesis of secondary metabolites, and stress responses [22]. In addition, bHLH plays an important role in the synthesis of anthocyanins [23,24], and bHLH and myeloblastosis viral oncogene homolog (MYB) transcription factors can promote pigment synthesis in Arabidopsis seedlings [25]. In apples, the MdbHLH3 transcription factor has also been shown to promote anthocyanin accumulation in fruits [26]. The WD repeat (WD40) / basic helix–loop–helix (bHLH)/ V‐myb avian myeloblastosis viral oncogene homolog (MYB) complex activates downstream signaling cascade under jasmonic acid (JA) induction, thereby regulating anthocyanin accumulation [27]. In addition, bHLH also plays an essential role in the synthesis of anthocyanins in gentian flowers [28]. In the color mutant jujube cultivar ‘Tailihong’ (TLH), the red color is due to anthocyanins [29]. Many studies have shown that bHLH transcription factors are involved in numerous physiological and biochemical processes in other plants. However, the molecular mechanisms of pigment formation in jujube fruit have not yet been elucidated, nor has the bHLH transcription factor family of jujube been fully identified. Therefore, our aim was to identify and analyze the bHLH transcription factors in the jujube genome and to investigate their changing expressions during fruit development. Our aim is that these findings will deepen our understanding of the functions of the jujube bHLH (ZjbHLH) genes in regulating the anthocyanin synthesis mechanism in jujube fruit. 2. Materials and Methods 2.1. Materials Two jujube cultivars, Ziziphus jujuba Mill. ‘Junzao’ (JZ) and ‘Tailihong’ (TLH), were obtained from the Jujube Experimental Station of Northwest Agriculture and Forestry University in Qingjian, Shaanxi, China. A total of 20 fruit samples of each genotype from three jujube trees were harvested at six developmental stages (S1…S6) on days 30, 50, 80, 90, 100 and 110 after anthesis (DAA), respectively (see Figure S1). The fruit skins (about 2 mm thick) were removed with a domestic vegetable peeler randomly. Composite skin samples were immediately frozen in liquid nitrogen and held at −80 ℃ pending analysis. 2.2. Extraction and Analyses of Total Anthocyanins Samples (about 0.5 g) of jujube fruit skin were extracted with 5 mL of 0.1% HCl in methanol for 24 h, at 4 ℃ in the dark. After centrifuging at 12,000 rpm for 15 min, three replicates were used for each sample [29]. The total anthocyanin content (TAC) was measured by the pH differential method. Absorbance was measured at 510 and 700 nm in buffers at pH 1.0 and at 4.5. The TAC is expressed as cyanidin‐3‐glucoside equivalents. 2.3. Total RNA Extraction and cDNA Synthesis The total RNA of all samples was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, Beijing, China) according to the manufacturer’s instructions. The purity and concentration of the extracted total RNA were determined using NanoDrop 20000 (Thermo Scientific, Forests 2019, 10, 346 3 of 12 Pittsburgh, PA, USA). Samples with an OD260/280 ratio of 1.8 to 2.0 and an OD260/230 ratio greater than 2.0 were retained. Subsequently, total RNA was reverse transcribed to obtain first strand cDNA. 2.4. Real‐time Quantitative Polymerase Chain Reaction (RT‐qPCR) Analysis Primers for real‐time Quantitative PCR (RT‐qPCR) were designed using Primer Premier 7.0 software (Premier, Palo Alto, CA, USA) (Table 1). The relative expression levels of target genes were detected using total RNA from different developmental stages of the two jujube cultivars as templates in CFX96 Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA). ZjUBQ and ZjUBQ2 were used as reference genes to correct the data [30]. The reaction system was 10 μL, including 1 μL cDNA, 1 μL each of the upstream and downstream primers, 5 μL of 2 × SYBR Premix Ex Taq II (TaKaRa) and 2 μL of ddH2O. The PCR reaction procedure was 95 ℃ for 10 s, 58 ℃ for 30 s, 72 ℃ for 45 s, 35 cycles of the above three steps and 72 ℃ for 5 min to complete the reaction. Table 1. RT‐qPCR primers. Gene name F (Primer sequence (5ʹ‐3ʹ)) R (Primer sequence (5ʹ‐3ʹ)) Length (bp) ZjGL3a GCATTCTGCTGCATTGTCTC CCCCTTTTTGCCTTTATTTT 194 ZjGL3b CAGCCACACCCAACCACTA CACCACACCTCCCAGAAAG 279 ZjTT8 ATCATCACACCCGCACAGAA CCCAACCAAAAGAGAACCCA 114 ZjUBQ TGGATGATTCTGGCAAAG GTAATGGCGGTCAAAGTG 98 ZjUBQ2 CACCCGTTACTTGCTTTC CTCTTCCCATTGTCCTCC 93 2.5. Sequence Bioinformatics Analysis Phylogenetic tree analysis of the bHLH transcription factor family obtained was generated using MEGA 7.0 (Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA). The conserved domain of the bHLH transcription factor family protein sequence was analyzed using DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA) and the online website WebLogo 3 (http://weblogo.berkeley.edu/logo.cgi). At the same time, their motif domains were predicted through the meme‐suite website (http://meme‐suite.org/). Chromosome localization

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